Interpenetrating microstructures for nanochannel-based sampling and/or cargo delivery

ABSTRACT

Provided herein are devices and methods for topically and controllably delivering cargo or collecting samples into or from biological tissues, particularly the skin. These devices permit delivery of cargo and collection of samples from cell layers deep within a tissue. These devices include microstructure arrays comprising nanochannels. Also disclosed is a device comprising a one or more microstructure arrays encased in a frame.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending application Ser.No. 16/471,907, filed Jun. 20, 2019, which is a National Stage ofInternational Application No. PCT/US2017/067630, filed Dec. 20, 2017,which claims benefit of U.S. Provisional Application No. 62/438,256,filed Dec. 22, 2016, which is hereby incorporated herein by reference inits entirety.

BACKGROUND

A common technique for delivering therapeutic agents across or into abiological tissue is the use of nanochannel arrays. However, currentnanochannel-based delivery methods can only directly deliver cargo tothe outermost cell layer of a tissue, significantly limiting the actionrange of this technology. Thus, there is a need for nanochannel-baseddelivery methods that can deliver cargo and/or sample substances fromcells from cell layers deep within a tissue.

SUMMARY

Provided herein are devices and methods for topically and controllablydelivering cargo and/or collecting samples into or out of biologicaltissues, particularly the skin. These devices permit delivery of cargoto deeper cell layers of a tissue. These devices include microstructurearrays comprising nanochannels. Also disclosed is a device comprising aone or more microstructure arrays encased in a frame. In someembodiments, the substance is an extracellular material, such asinterstitial fluid or an intracellular material.

In some embodiments, the microstructure array includes a planarsubstrate with a top surface and a bottom surface, a reservoir in fluidcommunication with the top surface of the planar substrate, and aplurality of microstructures projecting from the bottom surface of theplanar substrate. Each of the plurality of microstructures comprises asolid body portion tapering from a base to a distal tip positioned at aheight from the bottom of the planar substrate, thereby defining amicrostructure surface. Each of the plurality of microstructures alsocomprises a first delivery/sampling channel extending from the topsurface of the planar substrate to a first channel opening within themicrostructure surface, thereby fluidly connecting the reservoir to thefirst channel opening. Each of the plurality of microstructures alsocomprises a second delivery/sampling channel extending from the topsurface of the planar substrate to a second channel opening within themicrostructure surface, thereby fluidly connecting the reservoir to thesecond channel opening.

In some embodiments, the first channel opening is positioned within afirst plane parallel to the planar surface and the second channelopening is positioned within a second plane parallel to the planarsubstrate. In these embodiments, the first plane is distally spacedapart from the second plane. In some of these embodiments, the firstplane is distally spaced apart from the second plane by a distance offrom 20% to 60% of the height between the distal tip and the bottom ofthe planar substrate. In other embodiments, the first channel opening ispositioned at the distal tip.

In some embodiments, each of the plurality of microstructures furthercomprises a third delivery/sampling channel extending from the topsurface of the planar substrate to a third channel opening within themicrostructure surface, thereby fluidly connecting the reservoir to thethird channel opening. In some of these embodiments, the first channelopening is positioned at a first plane parallel to the planar substrate,the second channel opening is positioned within a second plane parallelto the planar substrate, the third channel opening is positioned withina third plane parallel to the planar substrate. In these embodiments thefirst plane is distally spaced apart from the second plane and thesecond plane is distally spaced apart from the third plane. In some ofthese embodiments, the first plane is distally spaced apart from thesecond plane by a distance of from 20% to 60% of the height between thedistal tip and the bottom of the planar substrate, and the second planeis distally spaced apart from the third plane by a distance of from 20%to 60% of the height between the distal tip and the bottom of the planarsubstrate.

In some embodiments the height between the distal tip and the bottom ofthe planar substrate is from 20 microns to 1000 microns.

In some embodiments the base has a substantially circular shape. Inother embodiments the base has a substantially rectangular shape.

In some embodiments the solid body portion is formed from silicon or asilicon-based material, e.g. silicon nitride. In other embodiments theplanar substrate is formed from silicon or a silicon-based material. Inother embodiments the solid body portion is formed from anodizedaluminum oxide. In other embodiments the planar substrate is formed fromanodized aluminum oxide.

In some embodiments, the planar substrate further comprises a pluralityof delivery channels, each of which extends from the top surface of theplanar substrate to a channel opening within the bottom surface of theplanar substrate, thereby fluidly connecting the reservoir to thechannel opening within the bottom surface of the planar substrate.

Also disclosed is a device comprising a one or more microstructurearrays encased in a frame. In some embodiments, the frame is pliableand/or malleable. In some cases, this allows for application to a curvedsurface. In some cases, the frame is non-planar. For example, the framecan be a cylinder such that the microstructure arrays arecircumferentially arranged around the cylinder. In some cases, amicrostructure array is positioned at the distal end of the cylinder. Insome of these embodiments, the cylinder defines a lumen, which canfunction as a reservoir for the microstructure arrays. In theseembodiments, an electrode can also be positioned in the lumen.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the fabrication of arrayedinterpenetrating nanochannels from silicon-based materials usingphotolithographic and etching techniques. First, an array of conical orpyramidal interpenetrating microstructures (approximately 20-500 micronstall) is defined on a silicon substrate using photoresist patterning andwet or dry etching. Subsequently, the silicon substrate is patterned onthe back-side with an array of nanowells (approximately 300-1000 nm indiameter) using projection lithography, which is then followed by ahighly anisotropic deep reactive ion etch (DRIE) to drill nanochannelsthrough the silicon substrate/interpenetrating microstructures.

FIG. 2 is a schematic diagram showing an alternative method offabricating arrayed interpenetrating nanochannels from silicon-basedmaterials using etching techniques. Selective surface etching is used todefine interpenetrating microstructures on a pre-made nanochanneledsubstrate platform.

FIG. 3 is a high resolution image showing one embodiment of amicrostructure array comprising nanochannels.

FIG. 4 is a schematic diagram showing the application of traditionalnanochannel-based delivery (e.g. TNT) methods.

FIG. 5 is a schematic diagram showing the delivery of cargo into/acrossa biological barrier using an interpenetrating microstructure arraycomprising nanochannels.

FIGS. 6a-n show TNT mediates enhanced reprogramming factor delivery andpropagation beyond the transfection boundary. FIG. 6a shows a schematicdiagram of the TNT process on exfoliated skin tissue. The positiveelectrode is inserted intradermally, while the negative electrode is putin contact with the cargo solution, A pulsed electric field (250 V, 10ms pulses, 10 pulses) is then applied across the electrodes tonanoporate exposed cell membranes and inject the cargo directly into thecytosol. Scanning electron micrographs (top) of the TNT platform surfaceshowing the nanopore array. FIG. 6b shows a schematic diagram showingthe boundary conditions for simulation purposes. FIG. 6c shows asimulation of the poration profile for different cells undergoing TNT(solid lines) vs. BEP (dashed lines). This plot shows that TNT leads tofocused poration, while BEP results in widespread poration. FIG. 6dshows ABM expression results for TNT vs. BEP. TNT resulted in superiorABM expression (t=24 hours), FIG. 6e shows representative IVISfluorescence and FIG. 6f shows confocal microscopy image of mouse skinafter TNT treatment with labeled DNA and the ABM factors, respectively.GFP is the reporter gene in the Ascl1 plasmid. FIG. 6g shows laserCapture Microdissection (LCM) and qRT-PCR results of gene expression inepidermis and dermis (t=24 hours) showing that gene expressionpropagated beyond the epidermal transfection boundary. FIG. 6h shows aschematic diagram illustrating the concept of EV-mediated transfectionpropagation from epidermis to dermis. FIG. 6i shows qRT-PCR analysis ofthe EV cargo showing significant loading of ABM mRNAs/cDNAs. FIG. 6jshows experimental design to confirm whether EVs are a viable vehiclefor propagating transfection and reprogramming. FIG. 6k shows confocalmicrograph showing a mouse embryonic fibroblast that has spontaneouslyinternalized the EVs isolated from TNT-treated skin. FIG. 6l shows geneexpression analysis 14 days after EV injection into naïve mice comparedto TNT-based transduction of ABM, Immunostaining results showingincreased (m) Tujl (week 4) and FIG. 6n shows neurofilament (week 8)expression in the skin after ABM TNT treatment. N=3 animals (biologicalreplicates). *p<0.05 (Holm-Sidak method), #0.05<p<0.08 (one-tailedt-test).

FIGS. 7a-k show TNT platform fabrication and nanochannel arraysimulation. FIG. 7a shows double side polished silicon wafer. FIGS. 7b-dshow nanochannel patterning and DRIE. FIG. 7e shows scanning electronmicroscopy (SEM) image of the etched nanochannels. FIG. 7f showsback-side etching of microreservoirs. FIG. 7g shows SEM micrographs andFIGS. 7h and 7i show plots showing etching profiles and etch rates,respectively, under different conditions. FIGS. 7j and 7k showsimulation results showing field distribution (j1, k1) and heatdissipation profiles 0, k 2-3) for asymmetric (i.e., T-shape)nanochannel arrays vs. symmetric (i.e., cross-shaped) arrays. Bulkelectroporation (BEP) is the current gold standard for non-viral genedelivery in vivo. Gene uptake in BEP, however, is a highly stochasticprocess, which is not only influenced by non-uniform electric fields,but also downstream and/or more passive processes such as endocytosisand diffusion, respectively (Geng, T. & Lu, C. Lab Chip 13, 3803-3821(2013). Boukany, P. E. et al. Nat Nanotechnol 6, 747-754 (2011);Gallego-Perez, D. et al. Nanomedicine (2015)). As such, simpleapproaches that facilitate more active and deterministic gene deliveryin vivo are clearly needed. Here cleanroom-based technologies wereimplemented (i.e., projection lithography, contact photolithography, anddeep reactive ion etching—DRIE—) (FIGS. 7a-i )) to fabricatesilicon-based TNT devices for active non-viral gene delivery tonaturally—(e.g., skin) or surgically-accessible (e.g., skeletal muscle)tissue surfaces in a more deterministic manner. The TNT platformsconsisted of a massively-parallel array of clustered nanochannelsinterconnected to microscale reservoirs that could hold the geneticcargo to be transduced into the tissues. Briefly, arrays of ˜400-500 nmchannels were first defined on the surface of a ˜200 μm thickdouble-side polished silicon wafer using projection lithography andDRIE. Simulation studies suggest that such asymmetric T-shape arrayprovides some inherent advantages in terms of electric fielddistribution and heat dissipation compared to a more symmetric nanoporedistribution, with asymmetric clusters of nanochannels exhibiting lessinactive zones (FIG. 7j 1, k1, stars), while at the same time reducingby 20-25% the peak and valley temperatures (FIGS. 7j 2-3, k2-3). Thiswas then followed by contact lithography-based patterning andDRIE-mediated drilling of an array of microreservoirs juxtaposing thenanochannels. Finally, the platform surface was passivated with a thininsulating layer of silicon nitride.

FIGS. 8a-h show simulation results of in vivo nanochannel-basedelectroporation vs. bulk electroporation (BEP). FIG. 8a shows aschematic diagram illustrating the experimental set-up. FIGS. 8b and 8cshow simulated voltage distribution under a 250 V stimulation. FIGS.8d-f show a simulation of transmembrane potential for single-cell bulkelectroporation. FIG. 8g shows a poration profile for a cell in directcontact with the nanochannel (cell 1) compared to cells far away fromthe nanochannels (cell 2 and cell 3). FIG. 8h shows profiles in TNT vs.BEP.

FIGS. 9a-d show autologous ABM-loaded EVs isolated from TNT-treateddorsal skin exhibit neurotrophic-like characteristics in a MCAO strokemouse (C57BL/6) model. FIG. 9a shows a schematic diagram illustratingthe experimental set-up. MCAO stroke is first induced. This is thenfollowed by ABM TNT treatment and EV isolation from dorsal skin prior tointracranial injection of EVs. FIGS. 9b and 9c show MRI imaging andquantification showing a significant reduction in the infarcted volumeonly 7 days after EV injection. FIG. 9d shows immunofluorescence imaging21 days after stroke induction showing DCX+ cells/processes projectingfrom the Subventricular (SVZ) zone towards the infarcted area (whitearrows). DCX+ cells in control brains were found mostly lining the wallsof the SVZ zone. *p<0.05 (Holm-Sidak method).

FIGS. 10a-b show iNs in the skin originate from epidermal and dermalsources. Fluorescence micrographs of ABM TNT-treated skin sections fromthe FIG. 10a shows K14-Cre reporter and FIG. 10b shows Col1A1-GFP mousemodels showing skin cells of either K14 or Col1A1 origin (green/GFP)also expressing the Tuj1 neuronal marker. (FIG. 10a .1, FIG. 10b .1)Cellular elements that were immunoreactive for both the GFP tracer andTuj1 were further analyzed by LCM/qRT-PCR. The results indicate thatsuch double-positive elements had significantly high neuronal markergene expression and moderate to markedly reduced skin cell marker geneexpression. *p<0.05 (Holm-Sidak method). Lineage tracing experimentswith a K14-Cre reporter mouse model, where Keratin 14 positive (K14+)cells undergo cre-mediated recombination of the ROSA locus ultimatelyswitching from tdTomato expression to eGFP, confirmed that thenewly-induced neurons partly originated from K14+ skin cells.Experiments with a Col1A1-eGFP mouse model, where cells with an activeCol1A1 promoter express eGFP, showed a number of Collagen/eGFP+ cellsfrom the dermis in a transition phase to Tuj1+. LCM was used to captureand further characterize the gene expression profile of cellularelements from the transgenic mouse model sections that were both GFP+and Tuj1+, which would correspond to cells that were of K14 origin butnow express a neuronal marker, or cells that have an active collagenpromoter (e.g., fibroblasts) transitioning to a neuronal fate. Theresults indicated that such elements indeed exhibited increasedexpression of pro-neuronal markers, and reduced expression of thecell-of-origin marker (i.e., K14, Col1A1).

FIG. 11 is illustration of a cross-sectional view of an examplemicrostructure embodiment.

FIG. 12 is illustration of a cross-sectional view of an examplemicrostructure embodiment.

FIG. 13 is illustration of a cross-sectional view of an examplemicrostructure embodiment.

FIGS. 14A-14D show various microstructure array arrangements andembodiments. FIG. 14A shows an embodiment with conical microneedles.FIG. 14B shows an embodiment with pyramidal microneedles. FIG. 14C showsan embodiment with concentrically-arranged cylinders. FIG. 14D shows anembodiment with ridges.

FIGS. 15A and 15B are plots comparing the relative level of geneexpression observed upon delivery of example reprogramming factors totissue using a TNT platform (FIG. 15A) and a DTN platform (FIG. 15B).Significantly higher levels of gene expression were observed in tissuewhen formulations were administered using the DTN platform.

FIG. 16 shows mouse skin tissue reprogramming into an endotheliallineage (i.e., CD31+ cellular structures, stained red) using the DTNplatform. A marked increase in CD31 staining across the entire thicknessof the tissue section was observed compared to control skin.

FIGS. 17A and 17B are illustrations of a cross-sectional view of anexample microstructure embodiment.

FIG. 18 shows various microstructure shapes.

FIG. 19 is a perspective view of a microstructure array embodimenthaving long slits in the body portion.

FIG. 20 is a perspective view of a microstructure array embodimenthaving short/arrayed slits in the body portion.

FIG. 21 is a cross-sectional view of the microstructure array embodimentof FIG. 20.

FIGS. 22A and 22B are perspective views of a microstructure arrayembodiment depicting reservoirs that can be machined into the bodyportion (FIG. 22A) or fabricated separately in another material and theninterfaced with the body portion (FIG. 22B).

FIG. 23 illustrates an embodiment microstructure viewed as across-section and its use for delivery of nucleic acid into skin tissueusing electroporation.

FIGS. 24A to 24D illustrate manufacturing of an embodimentsmicrostructure. FIG. 24A is a silicon wafer patterned via lithographyand deep reactive ion etching (DRIE) to create an array of nanochannels(FIG. 24B). These channels could range in size between ˜100-900 nm indiameter, with a pitch between edges ranging around 1-25 times thediameter. FIG. 24C illustrates the backside of the wafer being etched byDRIE to gain fluidic access to the nanochannels (thus creating a throughthickness array of nanochannels). FIG. 2D illustrates 2D array ofnanochannels being etched into 3D via lithography patterning andanisotropic etching. Such 3D array of nanochannels allows simultaneousand graded cargo delivery at multiple tissue levels/layers. The 3Dextrusions in FIG. 24D can range in size (at the base) between ˜1 andhundreds of microns, depending on the number of nanochannels (and gapin-between) that need to be accommodated within/across it. In some casesthe minimum number of nanochannels per 3D extrusion is 2, and these canbe a different heights to be able to enable delivery of cargo atdifferent levels of the tissue. Both nanochannels and/or 3D extrusionscan be arranged into hexagonal close packing (HCP) arrays to maximizethe active area of the device. Once the 3D extrusion is defined, thesurface can be coated with an insulating layer, which could be siliconnitride or silicon dioxide. Additional coatings that could be applied toimprove durability include (with compatible seed layers): siliconcarbide, titanium nitride, aluminum titanium nitride, and zirconiumnitride among others.

FIG. 25A to 25D illustrate an alternative fabrication approach. FIG. 25Aillustrates a substrate with already defined nanochannels subjected to(FIG. 25B) surface micromachining (e.g., through a lithographicapproach, or micromilling, for example) to create 3D extrusions of suchnanochannel array (FIGS. 25C and 25D). In this case the base substratecould be made of DRIE′d silicon, or anodized alumina, or a tack-etchedpolymeric membrane (e.g. PET).

FIGS. 26A to 26E illustrate embodiments for application to large/curvedtissue surfaces. FIG. 26A shows that a processed DTN device can be dicedinto smaller modules. Such modules can be encased within a pliablepolymeric frame to enable application on large/curved tissue surfaces.The picture in FIG. 26B shows an array of 3×3 modules being applied on ahuman arm. FIG. 26B large scale DTN could be used to intervenelarge/preclinical animal models and clinical models. In this case anischemic skin flap of a pig was DTN'd with EFF, which induces theformation of (FIG. 26D) new vasculature that can (FIG. 26E) rescue theflapped skin from necrosis (left) by increasing perfusion to that area(right).

DETAILED DESCRIPTION Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicant desires that the following terms be given the particulardefinition as defined below.

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “about” and “approximately” are defined as being “close to” asunderstood by one of ordinary skill in the art. In one non-limitingembodiment the terms are defined to be within 10%. In anothernon-limiting embodiment, the terms are defined to be within 5%. In stillanother non-limiting embodiment, the terms are defined to be within 1%.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

The term “carrier” or “pharmaceutically acceptable carrier” means acarrier or excipient that is useful in preparing a pharmaceutical ortherapeutic composition that is generally safe and non-toxic, andincludes a carrier that is acceptable for veterinary and/or humanpharmaceutical or therapeutic use. As used herein, the terms “carrier”or “pharmaceutically acceptable carrier” encompasses can includephosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations and as described further below.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose” refer to the amount of a composition, such asglucose-modified insulin bound to a glucose-binding structure, that willelicit the biological or medical response of a tissue, system, animal,or human that is being sought by the researcher, veterinarian, medicaldoctor or other clinician over a generalized period of time. In someinstances, a desired biological or medical response is achievedfollowing administration of multiple dosages of the composition to thesubject over a period of days, weeks, or years.

The term “subject” or “recipient” is defined herein to include animalssuch as mammals, including, but not limited to, primates (e.g., humans),cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and thelike. In some embodiments, the subject is a human.

The terms “treat,” “treating,” “treatment,” and grammatical variationsthereof as used herein, include partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition and/or alleviating,mitigating or impeding one or more causes of a disorder or condition.Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially.

DETAILED DESCRIPTION

The microstructure array and methods of using the same disclosed hereinare useful in transport of material into or across biological barriers.The microstructure array disclosed herein has the ability to deliversubstances to different layers of cells within tissue simultaneously (orsequentially) because the microstructures comprise multiple channelswhich can reach different levels of cells. This is because multiplechannels on an angled microstructure allow for different heights of thechannels within the microstructure. When the microstructure penetratestissue, the channels are placed within different layers of cells, andcan therefore deliver substances at different levels within the tissue.The microstructure array can be used on the skin (or parts thereof);across the blood-brain barrier; mucosal tissue (e.g., oral, nasal,ocular, vaginal, urethral, gastrointestinal, respiratory); bloodvessels; lymphatic vessels; or cell membranes (e.g., for theintroduction of material into the interior of a cell or cells). Thebiological barriers can be in humans or other types of animals, as wellas in plants, insects, or other organisms, including bacteria, yeast,fungi, and embryos. The microstructure array can be applied to tissueinternally with the aid of a catheter or laparoscope. For certainapplications, such as for drug delivery to an internal tissue, thedevices can be surgically implanted.

Referring to FIG. 1, the microstructure array (100) can comprise: aplanar substrate (101) having a top surface (102) and a bottom surface(103); a reservoir (104) in fluid communication with the top surface ofthe planar substrate; and a plurality of microstructures (105)projecting from the bottom surface of the planar substrate, each of theplurality of microstructures comprising: a solid body portion (106)tapering from a base (107) to a distal tip (108) positioned at a heightfrom the bottom surface of the planar substrate, thereby defining amicrostructure surface; a first delivery channel (109) extending fromthe top surface of the planar substrate to a first channel opening (110)within the microstructure surface, thereby fluidly connecting thereservoir to the first channel opening; and a second delivery channel(111) extending from the top surface of the planar substrate to a secondchannel opening (112) within the microstructure surface, thereby fluidlyconnecting the reservoir to the second channel opening.

Each of the delivery channels can be the same or different in scale. Insome embodiments, the delivery channels are nanochannels, e.g. having aninner diameter of about 1 to about 999 nm. In some embodiments, thedelivery channels are microchannels, e.g. having an inner diameter ofabout 1 to about 999 μm. The size of the channel can be selected basedon the size of the agent to be delivered and/or flow dynamics needed fora given application.

The planar substrate (101) can comprise a plurality of delivery channels(109, 111 for example), each of which extends from the top surface ofthe planar substrate to a channel opening (110, 112 for example) withinthe bottom surface (103) of the planar substrate, thereby fluidlyconnecting the reservoir (104) to the channel opening within the bottomsurface of the planar substrate.

As can be seen in FIGS. 1-2, the microstructure array (100) can beconfigured such that the first channel opening (110) is positionedwithin a first plane parallel to the planar substrate (101), wherein thesecond channel opening (112) is positioned within a second planeparallel to the planar substrate (101), and wherein the first plane isdistally spaced apart from the second plane. There can also exist athird channel opening (114) connected to a third channel (113). Theplanes of each channel and its associated opening can be spaced atregular intervals from each other, or at different intervals. Forexample, each channel/channel opening can be spaced equally apart fromthe others. For example, the first and second channel openings can bedistally apart by a distance of from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% ofthe height of the microstructure. In one example, the first plane can bedistally spaced apart from the second plane by a distance of 20% to 60%.This distance can apply to third, fourth, fifth, and more channelopenings as well.

The microstructures (105) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or more channels. These channels can act as a conduit betweenthe reservoir and the channel openings. Therefore, the channels can bein fluid communication with the reservoir, such that material placed inthe reservoir can travel through the channel and be delivered at thechannel openings. The first channel opening (110) can be positioned atthe distal tip, with additional channel openings flanking the distaltip. For example, there can be three channel openings on the samemicrostructure, with one being at the distal tip, and an additional twochannel openings (112 and 114) on either side of the distal tip.Multiple channels within the microstructure (105) can run parallel toeach other, and can run parallel to the vertical plane of themicrostructure, as shown in FIGS. 1 and 2.

As can be seen in FIGS. 17A and 17B, the height of the solid bodyportion (106) of the microstructure (105) can be measured from thedistal tip (108) of the microstructure to the base (107) of themicrostructure, which can be located on the bottom surface (103) of theplanar substrate (101). The height of the microstructure surface can be5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 microns in length, or more, less, or any amountin between. In one example, the height of the microstructure can be from100 to 500 microns. As can be seen in FIG. 17A, the height of themicrostructure (105) is measured in a plane b which can runperpendicular to the plane of the planar substrate (101).

The base (107) of the microstructure can have any shape, such as atriangle, square, diamond, rectangular, oval, or circle. In a particularembodiment, the base has a circular shape. When the base is a circle,the width of the base (107) of the microstructure (105) can be measuredas the diameter of the base of the microstructure where it makes surfacecontact with the planar substrate (101). The plane a of the base (107)of the microstructure (105) can run parallel with the planar substrate(101), as seen in FIG. 17B. The width of the base can be 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 microns in length, ormore, less, or any amount in between. The width of the base can also bemeasured in relation to the height of the microstructure, so that, forexample, the width of the base of the microstructure can be 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, or 200% of the height, or more, less,or any amount in between.

The distal tip (108) of the microstructure can have any shape, and canbe pointed, rounded, slanted, flared, tapered, blunted or combinationsthereof, as can be seen in FIG. 14A to 14D. In one embodiment, themicrostructure is pointed.

The microstructures can be spaced from each other at either regularintervals, or randomly. If spaced regularly, the microstructures can be5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000or more microns apart from each other, as measured from centerline b.The solid body portion (106) of the microstructures can comprise anyshape, such as a ridge, a herringbone pattern, a waveform pattern,cones, or pyramids. Examples of cones and pyramids can be seen in FIGS.14A, 14B, and 18.

In some cases, the microstructures lack an ordered shape or pattern. Forexample, the disclosed microstructures could be produced bytailored/blank etching of a silicon surface to introducerandomly-distributed microstructures/roughness with sharp tips that canpenetrate tissues/living systems.

The delivery channels (109, 111) can have various shapes, such as acylinder, cuboid, or rectangular prism, for example. The opening of thedelivery channel (110, 112) can be circular, rectangular, oval, orsquare, and as stated above, each microstructure (105) can have multiplechannels, which can all have the same geometry, or can have shapes whichdiffer from each other within the same or different microstructures. Thedelivery channel can have a substantially circular shape formingsubstantially concentric channels.

The reservoir (104) can comprise any substance to be delivered to asubject through the microstructure channel (109, 111). Conversely,substances can be drawn through the microstructure channel and depositedin the reservoir. The reservoir (104) can be in fluid communication withthe microstructure opening (110,112) through the microchannel, which canrun through the microstructure. The reservoir can therefore comprise anysubstance for transdermal administration. In one embodiment, thereservoir (104) is attached to the top surface (102) of the planarsubstrate, said top surface (102) being opposed to a bottom surface(104) of the planar substrate (101), wherein the microstructures projectfrom the bottom surface.

As shown in FIGS. 22A and 22B, the reservoir can be integral with theplanar substrate (101) (FIG. 22A), or it can be fabricated separately,e.g. in another material, and then interfaced with the planar substrate(101) (FIG. 22B). The reservoirs can be sized to each feed a pluralityof the microstructure channels, or arrayed so that each one feeds asingle microstructure. For example, in some embodiments, themicrostructure array (100) comprises a single large reservoir, eithermachined into the planar substrate (101) or interfaced with the planarsubstrate (101).

The reservoir can comprise a release mechanism for releasing thesubstance to be delivered, thereby permitting the substance to betransported into and through the at least one channel of themicrostructure. The release mechanism may utilize a mechanical force orsheer force, which can be manually, by heat, a chemical reaction, anelectric field, a magnetic field, a pressure field, ultrasonic energy,tension, diffusion injection, osmosis, concentration gradient, vacuum,pressure, or a combination thereof. In one embodiment, the reservoir(104) can include a porous material, wherein the substance to beadministered is stored in pores of the porous material. In anotherembodiment, the reservoir is sealed. In one variation of thisembodiment, the microstructure array further includes at least onepuncturing barb extending from the first surface of the planarsubstrate, wherein the puncturing barb can be used to puncture thesealed reservoir.

The reservoir can, for example, comprise a feedback component, such thatvolume or amount of the substance to be transported across thebiological barrier can be altered based on the physiological signal. Thefeedback component can comprise an “on and off” switch, such that when asignal is detected, the reservoir can deliver a substance to therecipient, but when no signal is detected, no substance is delivered.Conversely, the detection of a signal can have the opposite effect,wherein the reservoir defaults to delivery of a substance to therecipient, unless a signal is detected, which causes the reservoir notto release a substance for delivery to the recipient. By way ofillustration, the feedback component can detect the presence of apathogen in the subject, and when the substance is detected, thefeedback component can allow for the release of an antibody from thereservoir.

In another example, the feedback component can detect changes in aphysiological signal, such as pH or temperature. The feedback componentcan comprise a “cut off value” such that when the pH or the temperaturechanges by a certain amount, or reaches a certain numerical value (a pHbelow 6.5, for example, or a temperature above 99.1, for example), thefeedback component allows for a change in the release of the substance,or the amount of the substance released, and subsequently administeredto the recipient.

The feedback component can also adjust the amount or volume of thesubstance released based on the amount of signal detected, so that agreater amount of signal detected can result in a greater amount ofsubstance released, or conversely, a greater amount of signal detectedcan result in a smaller amount of substance released.

The physiological signal detected can, in one aspect, be any substancepresent in the subject to which the microstructure array is beingadministered. For example, the physiological signal can be a biologicalsubstance or a drug. The substance can either occur naturally in therecipient, or can be a non-endogenous, or foreign, substance. In anotheraspect, the physiological response in the subject can comprisephysiological environment factors, including pH and temperature.Examples of physiological signals include, but are not limited to,glucose, cholesterol, bilirubin, creatine, metabolic enzymes,hemoglobin, heparin, clotting factors, uric acid, carcinoembryonicantigen or other tumor antigens, reproductive hormones, oxygen, pH,temperature, alcohol, tobacco metabolites, and illegal drugs.

The substance in the reservoir to be delivered to the recipient can be atherapeutic, prophylactic, diagnostic, or theranostic substance. Morethan one substance can be delivered at a time, or different substancescan be delivered sequentially. Different substances can be deliveredthrough different channels at the same time, for example. 2, 3, 4, 5, 6,or more substances can be delivered simultaneously through differentchannels. Because the channel openings can reach different layers ofcells, different substances can be administered to different strata ofcells within tissue simultaneously utilizing the microstructure arraydisclosed herein. Specifically a first substance can be delivered via afirst channel to a first layer of cells, and a second substance can bedelivered via a second channel to a second layer of cells.

The substance to be delivered can be selected from the group consistingof peptides, proteins, carbohydrates, nucleic acid molecules, lipids,organic molecules, biologically active inorganic molecules, andcombinations thereof. For example, a wide range of drugs may beformulated for delivery with the present microneedle devices andmethods. As used herein, the terms “drug” or “drug formulation” are usedbroadly to refer to any prophylactic, therapeutic, diagnostic, ortheranostic agent, or other substance that which may be suitable forintroduction to biological tissues, including pharmaceutical excipientsand substances for tattooing, cosmetics, and the like. The drug can be asubstance having biological activity. The drug formulation may includevarious forms, such as liquid solutions, gels, solid particles (e.g.,microparticles, nanoparticles), or combinations thereof. The drug maycomprise small molecules, large (i.e., macro-) molecules, or acombination thereof. In representative, not non-limiting, embodiments,the drug can be selected from among amino acids, vaccines, antiviralagents, gene delivery vectors, interleukin inhibitors, immunomodulators,neurotropic factors, neuroprotective agents, antineoplastic agents,chemotherapeutic agents, polysaccharides, anti-coagulants, antibiotics,analgesic agents, anesthetics, antihistamines, anti-inflammatory agents,and viruses. The drug may be selected from suitable proteins, peptidesand fragments thereof, which can be naturally occurring, synthesized orrecombinantly produced. In one embodiment, the drug formulation includesinsulin.

The drug formulation may further include one or more pharmaceuticallyacceptable excipients, including pH modifiers, viscosity modifiers,diluents, etc., which are known in the art.

The reservoir disclosed herein can comprise the substance for releaseitself, or a means for producing a substance to be transported acrossthe biological barrier reservoir. One example of a means for producing asubstance is cells. The cells can be mammalian cells, such as humancells, or can be cells from any other source, which are capable ofproducing a substance for administration to a recipient. For example,the cells can pancreatic β cells or stem cell-differentiated humanpancreatic cells.

The substance, or the means for producing the substance, can be disposedin a reservoir which is semi-permeable, for example. This can allow forthe exchange of fluid with the recipient, such that the feedbackcomponent can be in fluid communication with the recipient, and therebydetect changes in physiological signal of the recipient. For example,the reservoir can comprise cells, wherein the cells are sensitive tochanges in a physiological signal from the recipient. Such physiologicalchanges in the recipient can stimulate the cells to release a substance,or to stop releasing a substance, as described above in regard to thefeedback component. In one example, the reservoir can comprise analginate microgel.

The microstructure array 100 disclosed herein can further include astimulus (release mechanism) for translocating the cargo from thereservoir to through the channels. In some cases, this stimulus involvesformation electrical field (e.g. electroporation), in which a poratingelectric field is applied to disrupt/deform lipid membranes and allowintracellular cargo delivery. In some embodiments, applying a poratingelectric field across the system results in the deformation and/ordisruption of cellular membranes, which allows for cargo translocationinto the intracellular space. Electric field strengths can beaccommodated depending on the target tissue/system. For example, FIG. 23illustrates use of the disclosed microstructure array 100 to delivernucleic acids into skin tissue using electroporation.

In other embodiments, the stimulus involves mechanical force, sheerforce, heat, chemical reaction, magnetic field, pressure field,ultrasonic energy, tension, diffusion injection, osmosis, concentrationgradient, vacuum, pressure, or a combination thereof. In someembodiments, the cargo comprises proteins, nucleic acids, or particles.However, in some embodiments, an electrical stimuli is the cargo beingdelivered. Pulsed electric fields have many applications, e.g. inregenerative medicine. The disclosed microstructure array 100 could beused to deliver pulsed electric fields at different levels across thetissue thickness.

Microneedles and electroporation apparatus are described in U.S. Pat.Nos. 6,334,856; 6,331,266; 6,312,612; 6,241,701; 6,233,482; 6,181,964,6,090,790; 6,014,584; 5,928,207; 5,869,326; 5,855,801; 5,823,993;5,702,359; 5,697,901; 5,591,139; 5,389,069; 5,273,525; and 7,127,284,which are incorporated by reference for this teaching.

The microstructure array can comprise a first electrode in electricalcontact with a second electrode, wherein the first electrode is incontact with the reservoir and the second electrode is in contact withone or more cells of a tissue while the microstructure array is in use.In order to enhance uptake of the substance, which may be a gene, othernucleic acid, protein or other large molecule, a small molecule drug, orthe like, an electric field can be established between electrodes spacedapart on opposite sides of the opening. The voltage, frequency, andother electrical field parameters will be selected primarily based onthe distance between the electrodes.

The microstructure array comprising electrodes can comprise a firstelectrode structure formed at the distal tip (108) of themicrostructure, such that it comes in contact with the cells and tissuethat have been punctured by the microstructure. The second electrodestructure can be disposed at the reservoir. The electrodes structurescan be formed as concentric bands that are connected to conductive pads.Each band and banded segment can be wired together to an electroporationpower supply, wired separately to an electroporation power supply, andcan be energized in a variety of geometric and timed patterns andarrangements. Moreover, the different bands and band segments can bemaintained at different electrical potentials (voltages) with respect tothe first electrode structure. A substance can be delivered through achannel opening (110, 112) at the distal tip (108) so that it permeatesthrough tissue outwardly in a region. The region can coincide with theelectrical field being generated between first electrode structure andsecond electrode structure. The electrical field enhances a cellularpermeability, thus enhancing the delivery of the desired targetsubstance to the cells.

The electroporation power supply can be a conventional power supply. Therequirements and specifications of such power supplies are welldescribed in the literature. See, for example, Neumann et al.,Electroporation and Electrofusion in Cell Biology, Plenum Press, NewYork, N.Y., 1989; Chang et al., Guide to Electroporation andElectrofusion, Academic Press, San Diego, Calif., 1992; Jaroszeski etal., Eletrochemotherapy, Electrogenetherapy, and Transdermal DrugDelivery: Electrically Mediated Delivery of Molecules to Cells, HumanaPress, Totowa, N.J., 2000; and Lynch and Davey, Electrical Manipulationof Cells, Chapman & Hall, New York, N.Y., 1996. The full disclosures ofeach of these publications are incorporated herein by reference.

The microstructure array capable of electroporation can comprise analternating current power supply adapted to deliver electroporationcurrent to the electrode structures at a desired voltage and frequency,typically selected to deliver electroporation current to the electrodesat a voltage in the range from 0.1 V to 30 kV. In some cases, thevoltage is less that about 300 to 500V. The particular voltage willdepend at least in part on the spacing between the first and secondelectrode structures. The frequency will typically be in the range from10 Hz to 107 Hz, usually from 104 Hz to 106 Hz. The current can beapplied at pulsed intervals, such as every 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more milliseconds, or anyamount in between. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more pulses can be applied in a given interval,and the intervals can be repeated until the desired result is achieved.

Also disclosed herein is a method for delivering one or more substancesto multiple cell levels of a tissue, comprising: providing amicrostructure array (100) comprising: a planar substrate (101) having atop surface (102) and a bottom surface (103); a reservoir (104) in fluidcommunication with the top surface of the planar substrate, wherein thereservoir comprises the substance to be delivered; and a plurality ofmicrostructures (105) projecting from the bottom surface of the planarsubstrate, each of the plurality of microstructures comprising: a solidbody portion (106) tapering from a base (107) to a distal tip (108)positioned at a height from the bottom surface of the planar substrate,thereby defining a microstructure surface; a first delivery channel(109) extending from the top surface of the planar substrate to a firstchannel opening (110) within the microstructure surface, thereby fluidlyconnecting the reservoir to the first channel opening; and a seconddelivery channel (111) extending from the top surface of the planarsubstrate to a second channel opening (112) within the microstructuresurface, thereby fluidly connecting the reservoir to the second channelopening; delivering the one or more substances within the reservoirthrough the delivery channels to multiple cell levels of the tissue.

Also disclosed herein is a method for delivering extracellular vesiclesfrom one layer of cells to another layer of cells, comprising: providinga microstructure array comprising: a planar substrate having a topsurface and a bottom surface; a reservoir in fluid communication withthe top surface of the planar substrate, wherein the reservoir comprisesthe substance to be delivered; and a plurality of microstructuresprojecting from the bottom surface of the planar substrate, each of theplurality of microstructures comprising: a solid body portion taperingfrom a base to a distal tip positioned at a height from the bottomsurface of the planar substrate, thereby defining a microstructuresurface; a first channel extending from the top surface of the planarsubstrate to a first channel opening within the microstructure surface,thereby fluidly connecting the reservoir to the first channel opening;and a second channel extending from the top surface of the planarsubstrate to a second channel opening within the microstructure surface,thereby fluidly connecting the reservoir to the second channel opening;isolating extracellular vesicles from a first layer of cells through thefirst channel; delivering the extracellular vesicles to a second layerof cells via a second channel. In one example, the first layer of cellscan be closer to the external surface than the second layer of cells.

The solid body portion of the microstructure array disclosed herein,including the planar substrate and reservoir, can be formed from avariety of materials, including silicon. Disclosed herein is a methodfor making the microstructure array (100). This can include the steps offorming a substantially planar substrate (101); and forming a pluralityof microstructures (105) projecting at an angle from the plane in whichthe planar substrate lies, the microstructures having a base (107)integrally connected to the substrate, a distal tip (108) connected tothe base, and body portion (106) therebetween, wherein at least one ofthe microstructures has at least one channel (109) extendingsubstantially from the base portion through at least a part of the bodyportion, the channel being open along at least part of the body portionand in fluid communication with the reservoir (104). In variousembodiments, the step of forming the microstructures comprisesembossing, injection molding, casting, photochemical etching,electrochemical machining, electrical discharge machining, precisionstamping, high-speed computer numerically controlled milling, Swissscrew machining, soft lithography, directional chemically assisted ionetching, or a combination thereof.

Also disclosed herein is a method is provided for administering asubstance to a subject in need thereof, which includes the steps ofinserting into the skin of the subject the microstructures (105) of thearray (100) described above, and causing the substance to be transportedfrom the reservoir (104) through the at least one channel of themicrostructure and through the stratum corneum of the skin.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1: Use of Tissue Nanotransfection Device for DirectCystolic Delivery of Reprogramming Factors

Disclosed herein is a device to topically and controllably deliverreprogramming factors to tissues through a nanochanneled device (FIG.6). Such tissue nano-transfection (TNT) approach allows, for example,the direct cytosolic delivery of reprogramming factors by applying ahighly intense and focused electric field through arrayed nanochannels(Gallego-Perez et al. Nanomedicine 2015; Boukany et al. Nat Nanotechnol2011, 6(11): 747-754), which benignly nanoporates the juxtaposing tissuecell membranes, and electrophoretically drives reprogramming factorsinto the cells (FIG. 6 a-d). A schematic of the TNT system fabricationprocess and simulation results can be found in FIGS. 1 and 2. Incontrast to current in vivo transfection technologies (e.g., viruses,conventional tissue bulk electroporation or BEP), in which gene deliveryis highly stochastic in nature and could lead to adverse side-effects(e.g., inflammatory response, cell death) (Sen C K et al. Am J Pathol2015, 185(10): 2629-2640), nanochannel-based delivery enables moreample, benign, instantaneous and dose-controlled reprogramming factordelivery at the single cell level, thus making this a safer and moredeterministic approach for in vivo gene transfection and reprogramming.

Experiments with FAM-labeled DNA on C57BL/6 mice established that TNTcan deliver cargo into the skin in a rapid (<I second) andnon-invasive/topical manner (FIG. 6e ). Next, it was determined thatTNT-based topical delivery of reprogramming factors can lead tosuccessful skin reprogramming using a robust model where overexpressionof Ascl1/Brn2/Myt1l (ABM) is known to directly reprogram fibroblastsinto induced neurons (iNs) in vitro (Vierbuchen et al. Nature 2010,463(7284): 1035-1041). These findings showed that TNT not only can beused for topical delivery of reprogramming factors (FIG. 6f ), but itcan also orchestrate a coordinated response that results inreprogramming stimuli propagation (i.e., epidermis to dermis) beyond theinitial transfection boundary (i.e., epidermis) (FIG. 6g-i ) possiblyvia dispatch of extracellular vesicles (EVs) rich in target genemRNAs/cDNAs (FIG. 6h,i ) (Valadi et al. Nat Cell Biot 2007, 9(6):654-659). Exposing naïve cells to ABM-loaded EVs isolated fromTNT-treated skin (FIG. 6j -1) established that these EVs can bespontaneously internalized by remote cells (FIG. 6k ). Moreover, geneexpression analysis indicated that intradermal ABM EV injectiontriggered changes in the skin consistent with neuronal induction (FIG.6l ), as evidenced by the approximately 25-fold increase in Tuj1expression compared to control skin. Comparatively, ABM-TNT resulted inan approximately 94-fold increase in Tuj1 expression, which reflects thenet effect of direct reprogramming factor injection combined withEV-mediated propagation. The neurotrophic effect of skin-derivedABM-loaded EVs was further confirmed in a middle cerebral arteryocclusion (MCAO) stroke mouse model (FIG. 9) (Khanna et al. J CerebBlood Flow Metab 2013, 33(8): 1197-1206).

Successful skin cell reprogramming was verified by immunofluorescence,which showed increased Tuft and Neurofilament expression overtime (FIG.6m,n ). Lineage tracing experiments with a K14-Cre reporter mouse modelconfirmed that the newly-induced neurons partly originated from K14+skin cells (FIG. 10). Hair follicles also consistently showed markedTujl immunoreactivity, suggesting that follicular cells couldpotentially play a role in the reprogramming process (Hunt et al. StemCells 2008, 26(1): 163-172; Higgins et al. J Invest Dermatol 2012,132(6): 1725-1727). Additional experiments with a CollAl-eGFP mousemodel (FIG. 10), where cells with an active CollAl promoter (e.g.,dermal fibroblasts) express eGFP, showed a number of Collagen/eGFP+cells in the dermis in a transition phase to Tujl+, thus suggesting alsoa fibroblastic origin for a proportion of the reprogrammed cells in theskin.

Therefore, it has been demonstrated herein that TNT can be used todeliver reprogramming factors into the skin in a rapid, highlyeffective, and non-invasive manner. Such TNT delivery leads to tailoredskin tissue reprogramming, as demonstrated with well-established andnewly developed reprogramming models of iNs and iECs, respectively.TNT-induced skin-derived iECs rapidly formed blood vessel networks thatsuccessfully anastomosed with the parent circulatory system and restoredtissue and limb perfusion in two murine models of injury-inducedischemia. This simple to implement TNT approach, which elicits andpropagates powerfully favorable biological responses through a topicalone-time treatment that only lasts seconds, can find applications in thedevelopment of novel interventional cell-based therapies for a widevariety of applications.

Methods

TNT Platform Fabrication

TNT devices were fabricated from thinned (approximately 200 μm)double-side polished (I00) silicon wafers (FIG. 7). Briefly,approximately I.5 μm thick layers of AZ5214E photoresist were first spincoated on the silicon wafers at approximately 3000 rpm. Nanoscaleopenings were subsequently patterned on the photoresist using a GCA6I00C stepper. Up to I6 dies of nanoscale opening arrays were patternedper I00-mm wafer. Such openings were then used as etch masks to drillapproximately 10 μm deep nanochannels on the silicon surface using deepreactive ion etching (DRIE) (Oxford Plasma Lab I00 system). Optimizedetching conditions included SF6 gas: I3 s/I00 sccm gas flow/700O W ICPpower/40 W RF power/30 mT APC pressure; C4F8 gas condition: 7 s/I00 sccmgas flow/700 W ICP power/I0 W RF power/30 mT APC pressure. Microscalereservoirs were then patterned on the back-side of the wafers viacontact photolithography and DRIE. Finally, an approximately 50 nm thickinsulating/protective layer of silicon nitride was deposited on the TNTplatform surface.

Animal Husbandry

C57BL/6 mice were obtained from Harlan Laboratory.B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice obtained fromJackson laboratories were bred with K14cre to produceK14cre/Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo/J mice. All mice weremale and 8-12 weeks old at the time of the study. Genotyping PCR forROSAmT/mG mice was conducted using primers oIMR73I8-CTC TGC TGC CTC CTGGCT TCT (SEQ ID NO: 1), olMR7319-CGA GGC GGA TCA CAA GCA ATA (SEQ ID NO:2) and olMR732O-TCA ATG GGC GGG GGT CGT T (SEQ ID NO: 3), while K-I4 Cretransgene was confirmed using primers olMRIO84-GCG GTC TGG CAG TAA AAACTA TC (SEQ ID NO: 4); olMRIO85-GTG AAA CAG CAT TGC TGT CAC TT (SEQ IDNO: 5). The animals were tagged and grouped randomly using a computerbased algorithm.

Mammalian Cell Culture and In Vitro Reprogramming

Primary human adult dermal fibroblasts (ATCC PCS-201-012) werepurchased, mycoplasma-free and certified, directly from ATCC. No furthercell line authentication was conducted. These cells were expanded infibroblast basal medium supplemented with fibroblast growthkit-serum-free (ATCC PCS 201-040) and penicillin/streptomycin. E12.5-E14mouse embryonic fibroblasts (MEFs) were cultured in DMEM/FI2supplemented with I0% fetal bovine serum. Non-viral cell transfectionand reprogramming experiments were conducted via 3D NanochannelElectroporation (NEP). Briefly, the cells were first grown to fullconfluency overnight on the 3D NEP device. Subsequently, a pulsedelectric field was used to deliver cocktail of plasmids (0.05 μg/μl)into the cells consisting of a 1:1:1 mixture of Fli1:Etv2:Foxc2. Thecells were then harvested 24 hours after plasmid delivery, placed inEBM-2 basal medium (CC-3I56, Lonza) supplemented with EGM-2 MVSingleQuot kit (CC-4I47, Lonza), and further processed for additionalexperiments/measurements.

In Vivo Reprogramming

The areas to be treated were first naired 24-48 hours prior to TNT. Theskin was then exfoliated to eliminate the dead/keratin cell layer andexpose nucleated cells in the epidermis. The TNT devices were placeddirectly over the exfoliated skin surface. ABM or EFF plasmid cocktailswere loaded in the reservoir at a concentration of 0.05-0.1 μg/μl. Agold-coated electrode (i.e., cathode) was immersed in the plasmidsolution, while a 24G needle counter-electrode (i.e., anode) wasinserted intradermally, juxtaposed to the TNT platform surface. A pulsedelectrical stimulation (i.e., I0 pulses of 250 V in amplitude and aduration of 10 ms per pulse) was then applied across the electrodes tonanoporate the exposed cell membranes and drive the plasmid cargo intothe cells through the nanochannels. ABM plasmids were mixed at a 2:I:Imolar ratio.

MCAO Stroke Surgery and Analysis

Transient focal cerebral ischemia was induced in mice by middle cerebralartery occlusion (MCAO) was achieved by using the intraluminal filamentinsertion technique previously described (Khanna et al. J Cereb BloodFlow Metab 2013, 33(8): 1197-1206). MRI images were used to determineinfarct size as a percentage of the contralateral hemisphere aftercorrecting for edema.

Hindlimb Ischemia Surgery

Unilateral hind-limb ischemia was induced via occlusion and subsequenttransection of the femoral artery followed by transection (Limbourg etal. Nat Protoc 2009, 4(12): 1737-1746). Briefly, 8-I0 week mice wereanesthetized with I-3% isoflurane, placed supine under astereomicroscope (Zeiss OPMI) on a heated pad. The femoral artery wasexposed and separated from the femoral vein through an approximately Icm incision. Proximal and distal end occlusion were induced with 7-0silk suture, which was then followed by complete transaction of theartery. Finally, a single dose of buprenorphine was administeredsubcutaneously to control pain. Laser speckle imaging (MoorLDI-Mark 2)was conducted 2 hours post-surgery to confirm successful blood flowocclusion.

Isolation of Extracellular Vesicles (EVs)

EVs were isolated from 12 mm diameter skin biopsies that were collectedin OCT blocks and stored frozen for later use. Briefly, the blocks werethawed and washed with phosphate buffer saline (PBS) to eliminate theOCT. Following removal of the fat tissue with a scalpel, the skin tissuewas minced into approximately I mm pieces and homogenized with amicro-grinder in PBS. After centrifugation at 3000 g, an Exoquick kit(System Biosciences) was used at a 1:5 ratio (Exoquick:supernatant) toisolate EVs from the supernatant for I2 hours at 4° C. EVs wereprecipitated via centrifugation at I500 g for 30 min. Total RNA was thenextracted from pellet using the mirvana kit (Life technologies)following the recommendations provided by the manufacturer.

DNA Plasmid Preparation

ABM and EFF plasmids were prepared using plasmid DNA purification kit(Qiagen Maxi-prep, catalogue number 12161, and Clontech Nucleobondcatalogue number 740410). DNA concentrations were obtained from aNanodrop 2000c Spectrophotemeter (Thermoscientific). A list of plasmidDNA constructs and their original sources can be found Table 1.

TABLE 1 Plasmid cDNA Plasmid Construct Name Gene Insert BackboneBrn2-RFP Brn2 pCAGGs Myt1l-CFP Myt1l pCAGGs Ascl1-GFP Ascl1 pCAGGspIRES-ER71(HA)3 Etsvp71 (ER71) pIRES-hrGFP-2a pAd -HA-Fli1-IRES- HA-Fli1pAd-IRES-GFP hrGFP mFoxc2 mFoxc2 pCDNA3.0

LCM was performed using a laser microdissection system from PALMTechnologies (Bernreid, Germany). Specific regions of tissue sections,identified based on morphology and/or immunostaining, were cut andcaptured under a 20× ocular lens. The samples were catapulted into 25 μlof cell direct lysis extraction buffer (Invitrogen). Approximately1,000,000 μm² of tissue area was captured into each cap and the lysatewas then stored at −80° C. for further processing. qRT-PCR of the LCMsamples were performed from cell direct lysis buffer followingmanufacture's instruction. A list of primers can be found in Table 2.

TABLE 2 List of Primers Primer/probe Name Primer Sequence Ascl1_q_F5′-CGACGAGGGATCCTACGAC-3′ (SEQ ID NO: 6) Ascl1_q_R5′-CTTCCTCTGCCCTCGAAC-3′ (SEQ ID NO: 7) Brn2_q_F5′-GGTGGAGTTCAAGTCCATCTAC-3′ (SEQ ID NO: 8) Brn2_q_R5′-TGGCGTCCACGTAGTAGTAG-3′ (SEQ ID NO: 9) Myt1L_q_F5′-ATACAAGAGCTGTTCAGCTGTC-3′ (SEQ ID NO: 10) Myt1L_q_R5′-GTCGTGCATATTTGCCACTG-3′ (SEQ ID NO: 11) PECAM1_F5′-GGACCAGTCCCCGAAGCAGC-3′ (SEQ ID NO: 12) PECAM1_R5′-AGTGGAGCAGCTGGCCTGGA-3′ (SEQ ID NO: 13) VEGFR2_F5′-AGCGCTGTGAACGCTTGCCT-3′ (SEQ ID NO: 14) VEGFR2_R5′-CATGAGAGGCCCTCCCGGCT-3′ (SEQ ID NO: 15) EGFP-N5′-CCGTCCAGCTCGACCAG-3′ (SEQ ID NO: 16) EGFP-C5′-GATCACATGGTCCTGCTG-3′ (SEQ ID NO: 17) Cdh5_F5′-GTGCAACGAGCAGGGCGAGT-3′ (SEQ ID NO: 18) Cdh5_R5′-GGAGCCACCGCGCACAGAAT-3′ (SEQ ID NO: 19) m-K14_F5′-GCTGGTGCAGAGCGGCAAGA-3′ (SEQ ID NO: 20) m-K14_R5′-AGACGGCGGTAGGTGGCGAT-3′ (SEQ ID NO: 21) m-Tuj1_F5′-TACACGGGCGAGGGCATGGA-3′ (SEQ ID NO: 22) m-Tuj1_R5′-TCACTTGGGCCCCTGGGCTT-3′ (SEQ ID NO: 23) m-Col1A1_F5′-GTGTGATGGGATTCCCTGGACCTA-3′ (SEQ ID NO: 24) m-Col1A1_R5′-CCTGAGCTCCAGCTTCTCCATCTT-3′ (SEQ ID NO: 25) m-MAP2_F5′-AGGCCAGGTGGTGGACGTGT-3′ (SEQ ID NO: 26) m-MAP2_R5′-CACGCTGGACCTGCTTGGGG-3′ (SEQ ID NO: 27) m-GAPDH_F5′-GTGCAGTGCCAGCCTCGTCC-3′ (SEQ ID NO: 28) m-GAPDH_R5′-GCACCGGCCTCACCCCATTT-3′ (SEQ ID NO: 29)

Immunohistochemistry and Confocal Microscopy

Tissue immunostaining was carried out using specific antibodies andstandard procedures. Briefly, OCT-embedded tissue was cryosectioned atI0 μm thick, fixed with cold acetone, blocked with I0% normal goat serumand incubated with specific antibodies. Signal was visualized bysubsequent incubation with fluorescence-tagged appropriate secondaryantibodies (Alexa 488-tagged a-guinea pig, I:200, Alexa 488-taggeda-rabbit, I:200; Alexa 568-tagged a-rabbit, I:200) and counter stainedwith DAPI. Images were captured by laser scanning confocal microscope(Olympus FV I000 filter/spectral).

IVIS Imaging

The animals were imaged with anesthesia 24 h after FAM-DNA transfectionusing IVIS Lumina II optical imaging system. Overlay images withluminescence images were made using Living Image software.

Magnetic Resonance Imaging (MRI) of Stroked Brains

Magnetic resonance angiography was used to validate our MCAO model inmice and to optimize the occluder size and the internal carotid arteryinsertion distance for effective MCAO. T2-weighted MRI was performed onanesthetized mice 48 h after MCA-reperfusion using 9.4 T MRI (BrukerCorporation, Bruker BioSpin Corporation, Billerica, M A, USA). MR imageswere acquired using a Rapid Acquisition with Relaxation Enhancement(RARE) sequence using the following parameters: field of view (FOV)30×30 mm, acquisition matrix 256×256, TR 3,500 ms, TE 46.92 ms, slicegap 1.0 mm, rare factor 8, number of averages 3. Resolution of 8.5pixels per mm. Raw MR images were converted to the standard DICOM formatand processed. After appropriate software contrast enhancement of imagesusing Osirix v3.4, digital planimetry was performed by a masked observerto delineate the infarct area in each coronal brain slice. Infarct areasfrom brain slices were summed, multiplied by slice thickness, andcorrected for edema-induced swelling as previously described todetermine infarct volume (Khanna S, et al. J Cereb Blood Flow Metab2013, 33(8):1197-1206).

Analysis of Muscle Energetics

Muscle energetics was evaluated NMR spectroscopy measurements on a 9.4Tesla scanner (Bruker BioSpec) using a volume coil for RF transmissionand a 3IP coil for reception (Fiedler et al. MAGMA 2015, 28(5):493-501.). In vivo imaging was conducted in a custom-made 1H/31Ptransceiver coil array. Data were acquired using single pulse sequence.The raw data were windowed for noise reduction and Fourier transformedto spectral domain.

Ultrasound-Based Imaging and Characterization of Blood Vessels

Blood vessel formation was parallely monitored via ultrasound imaging.Briefly, a Vevo 2100 system (Visual Sonics, Toronto, ON, Canada) wasused to obtain ultrasound images on B-mode with a MS 250 linear arrayprobe (Gnyawali et al. J Vis Exp 2010(41). Doppler color flow imagingwas implemented to monitor and quantify blood flow characteristics undersystole and diastole.

Statistical Analysis

Samples were coded and data analysis was performed in a blinded fashion.For animal studies, data are reported as mean±SD of 3 animals (i.e.,biological replicates). No animals were excluded from the analysis.Likewise, in vitro reprogramming data are reported as mean±SD of atleast 3 experiments. Experiments were replicated at least twice toconfirm reproducibility. Comparisons between groups were made byanalysis of variance (ANOVA). Statistical differences were determinedvia parametric/non-parametric tests as appropriate with SigmaPlotversion 13.0.

The disclosed results using TNT-based approach are indicative of whatcan be accomplished using nanochannel-based delivery, including themicrostructure array disclosed herein.

Example 2

In a particular embodiment, arrayed interpenetrating nanochannels can befabricated from silicon-based materials using cleanroom methods such asphotolithography and wet or dry etching. First, an array of conical orpyramidal interpenetrating microstructures (approximately 20-500 micronstall) is defined on a silicon surface using photolithography and wet ordry etching. Subsequently, the silicon substrate is patterned on thebackside with an array of nanowells (approximately 300-1000 nm diameter)using projection lithography. Next a highly anisotropic deep reactiveion etch (DRIE) to drill nanochannels through the silicon substrate andthe interpenetrating microstructures.

The following is an example method for fabrication of nanochanneledmicroneedle arrays on silicon

(1) Fabrication of 3D Nanochannel Array

1. Select 4 inch silicon wafer: 500 μm, DSP, Prime grade.

2. Thin the wafer to 250 μm thick by wet etching (KOH, 45%, 80 DegreeC.)

3. Pattern AZ-5214 nano-circle array (400 nm in diameter, spacing: 25μm) using Stepper (NTW).

4. DRIE Etch the silicon wafer masked by AZ-5214 pattern.

System: Oxford Plasma Lab 100 in Dreese Lab

Recipe Name: Lingqian_Bosch

SF6 Protocol: 100 sccm/13 s/ICP 700 W/RF 30 W/Chamber Pressure 30 mT(double check)

C4F8 Protocol: 100 sccm/7 s/ICP 700 W/RF 10 W/Chamber Pressure 30 mT(double check)

Etching Procedure: Running 40 cycles; Stop 5 minutes; Running another 5cycles; Stop 5 minutes and check if there is still photoresist remained.If yes, run another 5 cycles. Check again. Usually, the PR are totallygone with 55-60 cycles. (Please be aware, don't directly run 60 cyclesinstead of 40+5+5+5+5. Otherwise, the PR will be burned after 60 cycles)

5. Clean the silicon wafer/AZ5214 completely using TMP; Clean the waferin Piranha solution for 5 minutes (120 Degree C.). Check if thenanochannel array is visible using Olympus Microscope in Bay 1 (NTW)

6. Standard Photolithography procedure for pattern microchannel array(50 μm in diameter; Center to Center distance: 70 μm) on the other sideof the silicon wafer. Photoresist: SPR 220-7; Thickness: 10 μm. In thepatterning procedure: Make sure the array of microchannel is generallywith same direction to the nanochannel array.

7. Prepare another silicon wafer with 250 μm in thickness.

8. Fully coat the pump oil (used for ETC04 in Bay 4, NTW) on the side ofthe nanochannel. Key: oil all over the surface of the silicon waferrather than a droplet. Otherwise, the area without oil will cook the SPR220 PR in DRIE procedure.

9. Carefully and fully bond the 250 μm thick silicon wafer withnanochannel side, interfaced with oil. This way is to reduce the heat inlong time DRIE system while keep silicon wafer rigid enough to thehelium force in DRIE procedure.

10. DRIE Etch the silicon wafer masked by SPR 220-7.

System: Oxford Plasma Lab 100 in Dreese Lab

Recipe Name: Lingqian_Bosch

SF6 Protocol: 100 sccm/13 s/ICP 700 W/RF 30 W/Chamber Pressure 30 mT(double check)

C4F8 Protocol: 100 sccm/7 s/ICP 700 W/RF 10 W/Chamber Pressure 30 mT(double check)

Etching Procedure: Running 250 cycles; Stop 5 minutes; Running another20 cycles; Stop 5 minutes and check if there is still photoresistremained. If yes, run another 10 cycles. Check again. In experience,microchannel will connect nanochannel after about 280 cycles. However,the precise cycle is impossible to fix as the Oxford Plasma system isalways drifting. Therefore, after 270 cycles, SEM should be used tocheck the cross-section of microchannel in any stop time, to evaluatethat if all microchannel has connected with nanochannel (This part isthe mostly time consuming, which need about 3-5 days, depending onavailability)

11. Slowly and carefully remove the sample from

12. Clean the wafer in Piranha solution for 5 minutes (120 Degree C.).Check if the nanochannel array and microchannel array using OlympusMicroscope in Bay 1 (NTW)

13. Depends on the design of nanochannel, the silicon wafer was dicedinto small pieces.

(2) Fabrication of Microneedles Using DRIE

1. Pick up one silicon sample with the dimension of 1 cm×1 cm andthoroughly clean the sample with piranha solution.

2. Pattern micro-circle array on the nanochannel side: SPR 220-7photoresist, 10 μm in thickness. Micro-circle pattern: 15 μm indiameter, which finally determined the dimension of the volcano-shapedneedle. Spacing: center-center 25 μm. Ideal Condition: alignmicro-circle with nanochannel array in photolithography, which canincrease the hollow needle percentage.

3. Stick small sample on a fresh 4 inch wafer with oil (full-area coatedwith oil)

4. DRIE Procedure: Oxford Plasma Lab 100

Recipe: Lingqian_Isotropic Etch (double check)

Protocol: SF6: 40 sccm/ICP power: 600 W/RF power 5 W-10 W (whichdetermine the shape of the volcano). Etching time: 2 mins; stop andcheck if there are PR remained; IF yes, 1 more minutes and check if PRis remained; If Yes, one more minutes. If gone, check the microscope orSEM. If needle is not shown, continue etching 1 minute without mask.

5. Under the optimized protocol above, the micro-needle will befabricated with ˜2+1+1 minutes.

Example 3

In one particular embodiment, arrayed interpenetrating nanochannels canbe fabricated from a premade nanochanneled substrate platform that caneither be silicon-based or made from anodized alumina via selectivesurface etching. Selective surface etching is used to defineinterpenetrating microstructures

Example 4

In a preferred embodiment, a method delivering cargo to biologicaltissues at multiple levels is performed using an interpenetratingnanochannel array. The interpenetrating nanochannel array is placed incontact with exfoliated skin tissue. A positive electrode is insertedintradermally, while a negative electrode is put in contact with thecargo solution. A pulsed electric field (250 V, 10 ms pulses, 10 pulses)is then applied across the electrodes to nanoporate exposed cellmembranes and inject the cargo directly into the cytosol.

The cargo solution comprised a mixture of three plasmids, encoding forEtv2, Foxc2 and F11. Small tissue biopsies were collected 24 h afterdelivery and analyzed by qRT-PCR. Additional biopsies were collected 7days post-delivery and analyzed via immunohistochemistry for endothelialmarkers. DTN-based delivery results in superior transgene expression aswell as widely distributed reprogramming responses across the entiretissue thickness

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

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What is claimed is:
 1. A method for obtaining a sample from cells withina tissue, the method comprising: a) providing a microstructure arraythat comprises: a planar substrate having a top surface and a bottomsurface; a reservoir in fluid communication with the top surface of theplanar substrate; and a plurality of microstructures projecting from thebottom surface of the planar substrate, each of the plurality ofmicrostructures comprising: a solid body portion tapering from a base toa distal tip positioned at a height from the bottom surface of theplanar substrate, thereby defining a microstructure surface; a firstsampling channel or slit extending from the top surface of the planarsubstrate to a first channel opening or slit within the microstructuresurface, thereby fluidly connecting the reservoir to the first channelopening or slit; and a second sampling channel or slit extending fromthe top surface of the planar substrate to a second channel opening orslit within the microstructure surface, thereby fluidly connecting thereservoir to the second channel opening or slit; and a first electrodein electrical contact with the reservoir and a second electrodeconfigured to electrically contact a tissue positioned against thebottom surface of the planar substrate, wherein the solid body portionof each of the plurality of microstructures is formed from silicon usingphotolithography and etching, wherein the first sampling channel or slitand second sampling channel or slit have an inner diameter of 1 to 999nm, wherein the first channel opening or slit of each of the pluralityof microstructures is positioned within a first plane parallel to theplanar substrate, wherein the second channel opening or slit of each ofthe plurality of microstructures is positioned within a second planeparallel to the planar substrate, and wherein the first plane isdistally spaced apart from the second plane, wherein the first plane isdistally spaced apart from the second plane by a distance of from 20% to60% of the height from the bottom surface of the planar substrate, andwherein the first channel opening or slit is positioned at the distaltip of each of the plurality of microstructures; b) applying themicrostructure array to the tissue, wherein the bottom surface of theplanar substrate is positioned against the tissue and the plurality ofmicrostructures extend into the tissue; and c) sampling a substance fromthe cells within a tissue through the first sampling channel and thesecond sampling channel of each of the plurality of microstructures tothe reservoir by applying an electric field through the first electrodeand second electrode.
 2. The method of claim 1, wherein the each of theplurality of microstructures further comprises a third sampling channelextending from the top surface of the planar substrate to a thirdchannel opening or slit within the microstructure surface, therebyfluidly connecting the reservoir to the third channel opening or slit.3. The method of claim 2, wherein the first channel opening or slit ispositioned within a first plane parallel to the planar substrate,wherein the second channel opening or slit is positioned within a secondplane parallel to the planar substrate, wherein the third channelopening or slit is positioned within a third plane parallel to theplanar substrate; and wherein the first plane is distally spaced apartfrom the second plane and the second plane is distally spaced apart fromthe third plane.
 4. The method of claim 1, wherein the height of themicrostructure surface is from 5 microns to 1000 microns, and whereinthe width of the base of the microstructure is from 5 microns to 500microns.
 5. The method of claim 1, wherein the base of themicrostructure has a substantially circular shape or rectangular shape.6. The method of claim 1, wherein the plurality of microstructurescomprise parallel ridges, ridges arrayed in a herringbone pattern,ridges arrayed in a waveform pattern, cones, or pyramids.
 7. The methodof claim 1, wherein the distal tip of the microstructure is pointed,rounded, slanted, flared, tapered, blunted or combinations thereof. 8.The method of claim 1, wherein the first sampling channel and the secondsampling channel each have a substantially circular or rectangularcross-section in a plane parallel to the planar substrate.
 9. The methodof claim 1, wherein the first sampling channel and the second samplingchannel each have a substantially toroidal cross-section in a planeparallel to the planar substrate; and wherein the second samplingchannel is coaxially disposed around the first sampling channel.
 10. Themethod of claim 1, wherein the first sampling channel has asubstantially circular cross-section in a plane parallel to the planarsubstrate and the second sampling channel has a substantially toroidalcross-section in a plane parallel to the planar substrate; and whereinthe second sampling channel is coaxially disposed around the firstsampling channel.
 11. The microstructure array of claim 1, wherein eachof the plurality of microstructures comprises a solid body portiontapering in a stepwise fashion from the base to the distal tip.
 12. Themethod of claim 1, wherein each of the plurality of microstructurescomprises a solid body portion having a frustoconical shape.
 13. Themethod of claim 1, wherein the planar substrate further comprises aplurality of sampling channels, each of which extends from the topsurface of the planar substrate to a channel opening within the bottomsurface of the planar substrate, thereby fluidly connecting thereservoir to the channel opening within the bottom surface of the planarsubstrate.
 14. The method of claim 1, wherein the substance issimultaneously sampled from multiple cell levels of the tissue.
 15. Themethod of claim 1, wherein the electric field is applied at a potentialof from 1 V to 500 V.
 16. The method of claim 15, wherein the electricfield comprises a pulsed electric field.
 17. The method of claim 16,wherein the pulsed electric field comprises a plurality of pulses havinga duration of from 0.1 millisecond to 100 milliseconds.
 18. The methodof claim 17, wherein the pulsed electric field comprises from 1 to 500pulses.
 19. The method of claim 1, wherein the substance comprises anextracellular material.
 20. The method of claim 19, wherein theextracellular material comprises interstitial fluid.
 21. The method ofclaim 19, wherein the extracellular material comprises intracellularmaterial.
 20. A method for delivering a substance to stem cell nicheswithin a skin tissue, the method comprising: a) providing amicrostructure array that comprises: a planar substrate having a topsurface and a bottom surface; a reservoir in fluid communication withthe top surface of the planar substrate; and a plurality ofmicrostructures projecting from the bottom surface of the planarsubstrate, each of the plurality of microstructures comprising: a solidbody portion tapering from a base to a distal tip positioned at a heightfrom the bottom surface of the planar substrate, thereby defining amicrostructure surface; a first delivery channel or slit extending fromthe top surface of the planar substrate to a first channel opening orslit within the microstructure surface, thereby fluidly connecting thereservoir to the first channel opening or slit; and a second deliverychannel or slit extending from the top surface of the planar substrateto a second channel opening or slit within the microstructure surface,thereby fluidly connecting the reservoir to the second channel openingor slit; and a first electrode in electrical contact with the reservoirand a second electrode configured to electrically contact a tissuepositioned against the bottom surface of the planar substrate, whereinthe solid body portion of each of the plurality of microstructures isformed from silicon using photolithography and etching, wherein thefirst delivery channel or slit and second delivery channel or slit havean inner diameter of 1 to 999 nm, wherein the first channel opening orslit of each of the plurality of microstructures is positioned within afirst plane parallel to the planar substrate, wherein the second channelopening or slit of each of the plurality of microstructures ispositioned within a second plane parallel to the planar substrate, andwherein the first plane is distally spaced apart from the second plane,wherein the first plane is distally spaced apart from the second planeby a distance of from 20% to 60% of the height from the bottom surfaceof the planar substrate, and wherein the first channel opening or slitis positioned at the distal tip of each of the plurality ofmicrostructures; b) applying the microstructure array to the tissue,such that the bottom surface of the planar substrate is positionedagainst the tissue and the plurality of microstructures extend into thetissue; and delivering a substance from the reservoir through the firstdelivery channel and the second delivery channel of each of theplurality of microstructures to the stem cell niches within the tissue.